Bilirubin is a dye that is present mostly in bile in a living body, and mainly is produced from hemoglobin when senescent red blood cells, whose principal component is hemoglobin, are decomposed and metabolized in the marrow, the thymus, the liver, and the like. Bilirubin produced from hemoglobin is bound with albumin in blood, and is called unconjugated (indirect) bilirubin. This unconjugated bilirubin is taken into the liver and conjugated with glucuronic acid, thereby becoming conjugated (direct) bilirubin, and is discharged to the bile duct. Unconjugated (indirect) bilirubin and conjugated (direct) bilirubin collectively are called total bilirubin. Generally most of total bilirubin is indirect bilirubin.
The clinical significance in the assay of bilirubin in a biological sample has been well known conventionally. For example, hepatocellular disorder, intrahepatic cholestasis, extrahepatic cholestasis, or the like can be predicted from an increase in a direct bilirubin amount, while an increase in the amount of produced bilirubin, liver function abnormality, or the like can be predicted from an increase in an indirect bilirubin amount. As described above, the total bilirubin amount is an amount of a sum of the direct bilirubin amount and the indirect bilirubin amount. Therefore, if the total bilirubin amount and the direct bilirubin amount are known, the indirect bilirubin amount can be determined.
As methods for assay of bilirubin, the diazo method, the vanadate method, the enzyme method, the high precision liquid chromatography (HPLC) method, and the like are known. The enzyme method is a method in which an enzyme such as a bilirubin oxidase is allowed to act on a bilirubin-containing sample so as to oxidize bilirubin into biliverdin, whereby the light absorption by bilirubin (maximum absorption wavelength: 450 nm and the vicinity of the same) is eliminated, and a concentration of bilirubin is determined based on a decrease in the absorbance.
In the case where the total bilirubin is assayed by the enzyme method, there are problems in that the reaction with a bilirubin oxidase takes time or the reaction is insufficient, owing to the binding of indirect bilirubin with albumin. To cope with these problems, a surfactant or another substance is added as a reaction accelerator, which has been disclosed (see Patent Documents 1 to 3).
Patent Document 1 discloses a liquid-system method for assaying bilirubin in which first of all a sample containing bilirubin is mixed with a buffer solution containing sodium cholate, sodium dodecyl sulfate (SDS), or the like as an agent for transforming bilirubin into a direct type, and thereafter an enzyme reagent containing a bilirubin oxidase is added to the mixture. Patent Document 2 discloses a liquid-system method for assaying bilirubin in which a sample containing bilirubin is added to a bilirubin oxidase solution containing sodium cholate, SDS, or the like as a reaction accelerator. Patent Document 3 discloses a liquid-system method for assaying bilirubin in which a bilirubin oxidase and a sample containing and bilirubin are allowed to react with each other in the presence of hydroxypyridine derivative or mercaptopyridine derivative for accelerating reaction.
Patent Document 1: JP 59 (1984)-130198 A
Patent Document 2: JP 62 (1987)-282598 A
Patent Document 3: JP 2006-34178 A